1. Field of the Invention
The present invention relates to novel N-acetyl-.beta.-D-glucosamine derivatives, a process for producing the same, reagents for determining N-acetyl-.beta.-D-glucosaminidase activity containing the derivatives as effective ingredients, and a method for determining N-acetyl-.beta.-D-glucosaminidase activity efficiently and accurately by using the derivatives.
2. Description of the Prior Art
N-Acetyl-.beta.-D-glucosamidase (hereinafter simply referred to as NAGase) is one of the enzymes in lysosomes distributed in the kidney tubular epithelium in large quantities, and participates in decomposition of glucoproteins and mucopolysaccharides. It is recognized that the amount of urinary NAGase increases in various renal diseases such as acute renal deficiency, glomerulonephritis, etc. or in post-operative kidney. It is also recognized that in the case of diabetes the amount of NAGase increases not only in urine but also in serum. As an aid for diagnosis and course observation of various such renal diseases and also as an index in studies on renal toxicity of drugs, the determination of NAGase activity has attracted much attention both in clinical fields and in animal experiments.
Substrates for use in determining NAGase activity hitherto known include, for example, p-nitrophenyl-N-acetyl-.beta.-D-glucosaminide [Methods Enzymol., 28, 702 (1972)], 4-methylumbelliferyl-N-acetyl-.beta.-D-glucosaminide [Clinica. Chimica. Acta., 24, 189 (1969)]and m-cresolsulfonephthaleinyl-N-acetyl-.beta.-D-glucosaminide [Clin. Chem., 29, 1713 (1983)].
However, the use of these compounds as the substrate for determining NAGase activity has a drawback in that aglycone formed by the action of enzyme must be determined in such a highly alkaline pH region of the reaction solution as about 10 to 11, so that the determination of enzyme activity can be made only by means of so-called end-point assay, in which the enzyme reaction is once discontinued to perform the determination of the enzyme activity, while the rate-assay method, which is the most suitable as the method for determining enzyme activity in most cases, cannot be used.
Recently, as substrates which can be used in said rate-assay method, in which enzyme activity is determined by directly measuring changes in absorbance while the enzyme reaction is in progress, there have been proposed 2-chloro-4-nitrophenyl-N-acetyl-.beta.-D-glucosaminide [Clin. Chem., 34, 2140 (1988)]and sodio-3,3'-dichlorophenolsulfonephthaleinyl-N-acetyl-.beta.-D-glucosaminid e [Japanese Patent Application Kokai (Laid-open) No. 63-309199].
However, these compounds have drawbacks in that neither of them can exhibit sufficient sensitivity at pH of 4.5-5.0, which is the optimum pH for NAGase, and further 2-chloro-4-nitrophenyl-N-acetyl-.beta.-D-glucosaminide cannot be dissolved in amounts necessary and sufficient for the determination of NAGase activity.
In this connection, some of the present inventors have lately proposed resorufinyl-N-acetyl-.beta.-D-glucosaminide or resazurinyl-N-acetyl-.beta.-D-glucosaminide as a substrate which can be effectively used in the rate-assay method (U.S. Pat. No. 5,030,721). However these substrates were not necessarily satisfactory in solubility in water.
The object of the present invention is to provide, overcoming the problems mentioned above involved in previous reagents for determining NAGase activity and in methods of determination using the reagents, a novel compound useful as a reagent which has an excellent solubility and enables effective and accurate determination of NAGase activity and also a novel method for determining NAGase activity using the compound.